Hepatitis A virus (HAV) is a picornavirus with a single-stranded RNA genome of approximately 7500 nucleotides. The wild-type strain of HAV grows poorly in cell-culture, generally is not cytopathic, and virus yields are low. A cell-culture adapted mutant has been selected which grows significantly more efficiently in cell-culture and which is attenuated for marmosets and chimpanzees. The objectives of this project are to determine the genetic basis for virulence and adaptation to cell culture of HAV in order to develop a strain of HAV suitable for use as an attenuated vaccine. The following advances in our understanding of HAV were made. In an effort to increase replicative capacity, chimeric viruses were constructed from two or more HAV strains including a virulent human strain, an attenuated strain, a vaccine strain, a cytopathic strain, and a simian strain. The P2 region from a cytopathic strain of HAV was shown to confer the large plaque phenotype but not the lytic phenotype of the cytopathic virus. We also constructed viable HAV mutants under the translational control of the encephalomyocarditis internal ribosome site. These chimeric viruses are being analyzed to determine if inclusion of this efficient control element enhances viral protein production or affects host range. Chimeric viruses were also constructed for the purpose of defining virulence genes. We identified the 2A gene as a second major HAV/7 determinant of attenuation for marmosets and obtained evidence that mutations in the 2A and 2C genes are almost totally responsible for the attenuation of the virus. A full length infectious cDNA of the attenuated HAV MRC-5 cell adapted vaccine strain was constructed to serve as a genetic repository for the vaccine strain and to permit detailed molecular analysis of the virus it encodes. Finally, evidence was obtained that mutations in the 5' non-coding region of the MRC-5 cell adapted virus attenuate the virus for marmosets.